The use of oligonucleotides and modified oligonucleotides is of great significance in modern therapy and has been well documented (Uhlmann, et al. Antisense oligonucleotides: A new therapeutic principle. Chemical Reviews 1990, 90: 543-584; Crooke, et al. “Antisense Research and Applications”, CRC Press (1993); Mesmaekar, et al. “Antisense oligonucleotides,” Acc. Chem. Res. 1995, 28: 366-374; Stein. “The experimental use of antisense oligonucleotides: a guide for the perplexed.” J. Clin. Invest. 2001, 108, 641-644.) The specific binding of antisense polynucleotides to the DNA or RNA targets can inactivate the replication, transcription, or translation of nucleic acids, thereby providing a mechanism for controlling diseases such as cancer and viral infection. The binding of an antisense oligonucleotide to a target can thus be used to alter gene expression, in a variety of circumstances, e.g., to interfere with viral life cycles, or the growth of cancerous cells.
In addition to specific binding affinity to a complementary target nucleotide sequence, antisense oligonucleotides should fulfill the requirements for therapeutic purposes, including potency, bioavailability, low toxicity, and low cost. Since oligonucleotides having a natural phosphodiester backbone are labile to nucleases and do not readily penetrate the cell membrane, researchers have attempted to make polynucleotide backbone modifications that improve nuclease resistance and cellular uptake. Therefore, it is desirable to provide polynucleotide analogs with enhanced nuclease resistance and cellular uptake, while retaining their specific interaction with nucleic acids and/or their catalytic activity.
Efforts have been directed to the development of chemical modifications of antisense oligonucleotides with higher resistance to nuclease activity (Mesmaekar, et al. “Antisense oligonucleotides.” Acc. Chem. Res. 1995, 28: 366-374; Crooke S T. “Progress in antisense therapeutics.” Med. Res. Rev. 1996; 16: 319-344). For instance, one approach (Wang, et al., “Sugar modified nucleosides and oligonucleotides” U.S. Pat. No. 5,681,940; Oct. 28, 1997) provides various novel sugar modified nucleosides and corresponding sugar modified oligonucleotides that have properties superior to natural RNA and DNA oligonucleotides when used for antisense, diagnostic, or other purposes. Various other modified nucleotides have been proposed as potential antisense drugs (Iyer, “Reagents and process for synthesis of oligonucleotides containing phosphorodithioate internucleoside linkages” U.S. Pat. No. 6,117,992; Sep. 12, 2000; Meyer, et al. “Oligonucleotides containing pyrazolo[3,4-d]pyrimidines for hybridization and mismatch discrimination” U.S. Pat. No. 6,127,121; Oct. 3, 2000; Froehler, et al. “Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines” U.S. Pat. No. 6,235,887; May 22, 2001; Cook, et al. “Substituted purines and oligonucleotide cross-linking” U.S. Pat. No. 6,232,463, May 15, 2001; Short, “Modified nucleotides and methods useful for nucleic acid sequencing” U.S. Pat. No. 6,579,704, Jun. 17, 2003).
An interesting approach to decrease the nuclease viability of oligonucleotides is via modification by inclusion of zwitterionic base forms, which electrostatically protects the phosphodiester bond (Switzer, “Antisense oligonucleotides comprising 5-aminoalkyl pyrimidine nucleotides” U.S. Pat. No. 5,596,091, Jan. 21, 1997; Switzer, “Antisense oligonucleotide containing compositions and method of forming duplexes” U.S. Pat. No. 6,031,086, Feb. 29, 2000).
It has been demonstrated by molecular modeling and accompanying experiments that certain modified purine and pyrimidine bases (e.g., 1-methyl-5-hydroxycytosine and its anionic form, 1-methyl-5-bromouracil, and 2-amino-9-methylpurine) possess zwitterionic tautomers that bind strongly with complementary native nucleic acid bases (Suen, et al. “Identification by UV resonance Raman spectroscopy of an imino tautomer of 5-hydroxy-2′-deoxycytidine, a powerful base analog transition mutagen with a much higher unfavored tautomer frequency than that of the natural residue 2′-deoxycytidine.” Proc. Natl. Acad. Sci. USA 1999, 96: 4500-4505. Karelson, et al. “Quantum-Chemical Modeling of the Tautomeric Equilibria of Modified Anionic Nucleic Acid Bases,” ARKIVOC, 2001, 3, 51-62.
There has also been great interest in designing organic-metal complexes that are capable of catalytically hydrolyzing nucleic acids. (Morrow, “Artificial Ribonucleases,” Adv. Inorg. Biochem., 1994, 9:41-74; Magda, “Metal Complex Conjugates of Antisense DNA Which Display Ribozyme-Like Activity,” J. Am. Chem. Soc. 1997, 119:6947-6948; Komiyama, “Progress towards synthetic enzymes for phosphoester hydrolysis,” Current Opinion in Chemical Biology, 1998, 2:751-757; Hegg, “Toward the development of metal-based synthetic nucleases and peptidases: a rationale and progress report in applying the principles of coordination chemistry,” Coord. Chem. Revs. 1998, 173: 133-165; Trawick et al. “Inorganic Mimics of Ribonucleases and Ribozymes: From Random Cleavage to Sequence-Specific Chemistry to Catalytic Antisense Drugs,” Chem. Rev. 1998, 98: 939-960.) The complexes of certain lanthanides (e.g., lanthanum, europium, cerium, gadolinium) possess comparable or even higher phosphodiesterase activity than the native enzymes (Bing Zhua, et al., “Binuclear lanthanide complexes as catalysts for the hydrolysis of double-stranded DNA,” Inorg. Chem. Communs., 1999, 2: 351-353; Williams, et al., “Structure and Nuclease Activity of Simple Dinuclear Metal Complexes: Quantitative Dissection of the Role of Metal Ions,” Acc. Chem. Res. 1999, 32: 485-493; Häner et al. “Development of Artificial Ribonucleases Using Macrocyclic Lanthanide Complexes,” Chimia 2000, 54:569-573; Kuzuya, et al., “Conjugation of Various Acridines to DNA for Site-Selective RNA Scission by Lanthanide Ion,” Bioconjugate Chem. 2002, 13: 365-369; Canaple, et al., “Artificial Ribonucleases: Efficient and Specific in Vitro Cleavage of Human c-raf-1 RNA,” Bioconjugate Chem. 2002, 13:945-951; Shigekawa et al., “Extended x-ray absorption fine structure study on the cerium(IV)-induced DNA hydrolysis: Implication to the roles of 4 f orbitals in the catalysis” Appl. Phys. Lett. 1999, 74: 460-462.)
One potential means of providing synthetic RNA transesterification catalysts may be via the creation of more potent antisense oligonucleotides through the attachment of a catalytic cleaving group, which would render them efficient and selective mutagenic and antiviral agents. (Hall, et al. “Efficient sequence-specific cleavage of RNA using novel europium complexes conjugated to oligonucleotides”, Chemistry & Biology, 1994, 1: 185-190; Komiyama, “Sequence-specific and hydrolytic scission of DNA and RNA by lanthanide complex-oligoDNA hybrids”, J. Biochem., 1995, 118:665-670; Hall et al. “Towards artificial ribonucleases: The sequence-specific cleavage of RNA in a duplex”, Nucl. Acid Res., 1996, 24: 3522-3526; Hall et al. “Sequence-specific cleavage of RNA using macrocyclic lanthanide complexes conjugated to oligonucleotides: A structure activity study,” Nucleosides & Nucleotides, 1997, 16: 1357-1368; Häner et al. “The sequence-specific cleavage of RNA by artificial chemical ribonucleases,” Antisense and nucleic acid drug development, 1997, 7: 423-430; Baker et al. “Oligonucleotide-europium complex conjugate designed to cleave the 5′ cap structure of the ICAM-1 transcript potentiates antisense activity in cells,” Nucl. Acid Res., 1999, 17:1547-1551; Haner et al, “Functional terpyridine-metal complexes, a process for the preparation thereof and oligonucleotide conjugates with terpyridine-metal complexes” U.S. Pat. No. 5,925,744, Jul. 20, 1999; Morrow, “Metal complexes for promoting catalytic cleavage of RNA by transesterification” U.S. Pat. No. 5,684,149; Nov. 4, 1997) Artificial enzymes for selective scission of RNA at one or two designated sites have been prepared by combining a lanthanide(III) ion with an oligonucleotide bearing one or two acridine groups. (Kuzuya et al. “Conjugation of Various Acridines to DNA for Site-Selective RNA Scission by Lanthanide Ion,” Bioconjugate Chem. 2002, 13: 365-369; Kuzuya et al. “Selective activation of two sites in RNA by acridine-bearing oligonucleotides for clipping of designated RNA fragments,” J. Am. Chem. Soc., 2004, 126: 1430-1436.)